Cellulose is the major structural polysaccharide of higher plant cell walls. Chains of β-1,4-linked glucosyl residues assemble soon after synthesis to form rigid, chemically resistant microfibrils. Their mechanical properties together with their orientation in the wall influence the relative expansion of cells in different directions and determine many of the final mechanical properties of mature cells and organs. These mechanical properties are of great importance for wood, paper, textile and chemical industries.
Much of the high quality fiber for the textile industry is provided for by cotton. About 90% of cotton grown worldwide is Gossypium hirsutum L., whereas Gossypium barbadense accounts for about 8%.
Several genes involved in cellulose biosynthesis have already been identified by mutational analysis in a number of plants. Mutants of Arabidopsis thaliana show that in vivo cellulose synthesis requires the activity of members of the AtCesA gene family encoding glycosyltransferases (Arioli et al., 1998; Taylor et al., 1999; Fagard et al., 2000; Taylor et al., 2000; Scheible et al., 2001; Burn et al., 2002a; Desprez et al., 2002), of the AtKOR1 gene (At5g49720) encoding a membrane-associated endo-1,4-β-D-glucanase (Nicol et al., 1998; Zuo et al., 2000; Lane et al., 2001; Sato et al., 2001), of KOBITO1 encoding a plasma membrane protein of unknown function (Pagant et al., 2002) and of genes encoding enzymes in the N-glycosylation/quality control pathway in the ER (Lukowitz et al., 2001; Burn et al., 2002b; Gillmor et al., 2002).
The function of an endo-1,4-β-D-glucanase in cellulose synthesis remains to be determined but the lack of activity against crystalline cellulose of BnCel16, a related Brassica napus enzyme (Mølhøj et al., 2001), suggests that the enzyme probably cleaves a non-crystalline glucan chain such as a lipid-linked primer or glucan donor (Williamson et al., 2001; Peng et al., 2002). Tomato Cel3 (LeCel3) was the first such membrane-associated endo-1,4-β-D-glucanase identified (Brummell et al., 1997) and antibodies to LeCel3 detected a cotton fiber protein upregulated during herbicide inhibition of cellulose synthesis (Peng et al., 2001). A cotton fiber membrane fraction required Ca2+ for in vitro cellulose synthesis activity and, because an exogenous, Ca2+-independent endo-1,4-β-D-glucanase restored cellulose synthesis activity, a cotton orthologue of KOR (GhKOR) was proposed as the endogenous Ca2+-dependent factor (Peng et al., 2002). A truncated form of BnCel16 showed Ca2+-dependence in vitro (Mølhøj et al., 2001).
Further genetic data point to cellulose synthesis responding to defects in enzymes on the N-glycosylation/quality control pathway. These steps occur in the ER rather than at the plasma membrane and so probably act only indirectly on synthesis through the supply of key glycoproteins to the plasma membrane. N-glycosylation begins when the mannose-rich oligosaccharide Glc3Man9GlcNac2 is assembled on dolichol in the ER membrane and transferred to the Asn residue of a newly synthesized protein containing an Asn-X-Ser or Asn-X-Thr motif (where X is any amino acid except Pro).
With further processing of the glycoprotein by glucosidases I and II, N-glycosylation intersects with the quality control pathway responsible for ensuring proper folding of newly synthesized proteins (Helenius and Aebi, 2001; Vitale, 2001). Glucosidase I removes the terminal α-1,2-linked glucosyl residue to generate Glc2Man9GlcNac2 and glucosidase II removes the next α-1,3-glucosyl residue. Polypeptides carrying the resultant GlcMan9GlcNac2 specifically bind chaperones (calnexin and calreticulin) and probably other proteins that promote proper folding of newly synthesized proteins. The glycoprotein releases the chaperones when glucosidase II trims of the final Glc residue which is required for chaperone binding. Glycoprotein glucosyltransferase then reattaches one Glc residue to the Man9GlcNAc2 of improperly folded glycoproteins so that they again bind chaperones and have a further opportunity to fold properly. Properly folded proteins, however, cannot be reglucosylated by that enzyme and progress though the secretory pathway for further processing and delivery.
Defects at several points in this pathway affect cellulose synthesis. Sequence analysis suggests that the potato MAL1 gene encodes a glucosidase II and antisense suppression reduces glucosidase II activity (Taylor et al, 2000a). M4LJ antisense plants accumulate less cellulose than controls when grown under field conditions although there is no visible phenotype in glasshouse conditions. The embryo lethal knopf mutant is deficient in glucosidase I and severely deficient in cellulose (Gillmor et al., 2002). Finally the embryo lethal cyt1 mutant is cellulose-deficient from a defect in mannose-1-phosphate guanylyltransferase, the enzyme generating the UDP-Man required to (amongst other things) assemble the high mannose oligosaccharide that is transferred from dolichol to the nascent protein (Lukowitz et al, 2001). The mutations that affect cellulose synthesis concentrate towards those early steps where the N-glycosylation pathway intersects with the quality control pathway. Quality control, rather than production of mature glycans on critical proteins, seems particularly important since there is no detectable phenotype from a defect in N-acetyl glucosaminyl transferase I that blocks the steps in the Golgi that build mature, N-linked glycans (von Schaewen et al, 1993).
Baskin et al. 1992 described Arabidopsis mutants which show root radial swelling, named rsw1, rsw2 and rsw3. These mutant lines where shown to exhibit a selective reduction in cellulose production (Peng et al. 2000).
WO98/00549 relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation, or as ribozymes, co-suppression or gene-targeting molecules. More particularly, this disclosure is directed to a nucleic acid molecule isolated from Arabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica spp. Gossypium hirsutum and Eucalyptus spp, which encode an enzyme which is important in cellulose biosynthesis, in particular the cellulose synthase enzyme and homologues, analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties.
WO 98/50568 discloses the use of a nucleotide sequence coding for an endo-1,4-β-glucanase to inhibit cell growth in a plant. The nucleotide sequence corresponds wholly or partially to the Arabidopsis KOR protein sequence, or to a protein sequence the N-terminal end of which has at least 40% identity with the first 107 amino acids of said KOR, or at least 70% identity with the first 107 amino acids of said KOR.
WO 97/24448 describes recombinant and isolated nucleic acids encoding a plant α-glucosidase enzyme. An antisense nucleotide was also provided as well as the use of both the isolated or recombinant sequences and the antisense sequences. Uses of the invention include enhancing and reducing expression of alpha-glucosidases and the provision of novel starches.
WO 00/08175 relates to nucleic acid molecules coding for a protein with the activity of an alpha-glucosidase from a potato. The invention also relates to methods for the production of transgenic plant cells and plants synthesizing modified starch. The invention further relates to vectors and host cells containing the nucleic acid molecules, plant cells and plants obtained according to the methods, starch synthesized by the described plant cells and methods for the production of such starch.
WO 98/39455 discloses a gene and enzyme participating in the synthesis of cellulose by microorganisms. A specific gene encoding a cellulase, cellulose synthase complex and alpha-glucosidase are described.
WO9818949 and U.S. Pat. No. 6,271,443 provide two plant cDNA clones that are homologs of the bacterial CelA genes that encode the catalytic subunit of cellulose synthase, derived from cotton (Gossypium hirsutum). Also provided are genomic promoter regions to these encoding regions to cellulose synthase. Methods for using cellulose synthase in cotton fiber and wood quality modification are also provided.
The prior art remains however deficient in providing alternatives to the known genes involved in cellulose biosynthesis and does not disclose the nucleotide sequence of the wild type gene involved in cellulose biosynthesis and mutated in the rsw3 mutant Arabidopsis line. Also, the prior art does not disclose the cotton homologues genes of RSW2 or RSW3 involved in cellulose biosynthesis from cotton.
These and other problems have been solved as set forth hereinafter in the different embodiments and claims of the invention.